For many years, researchers in the field of bioconjugate chemistry have needed well-defined ligation strategies that can be used for the at-will modification of biomolecules. Efficient bioconjugation strategies generally involve high levels of functional group tolerance, compatibility with water and other solvents, and efficient conversions (e.g., fast reaction times and high yields). Reactions that adhere to the principles of “click chemistry” (introduced by Sharpless and co-workers in 2001) are ideal candidates for bioconjugation applications. “Click” reactions are thermodynamically driven because the products have a highly favorable enthalpy of bonds. Several reactions can be classified as “click”, including copper-catalyzed Huisgen's dipolar cycloaddition of azides and terminal alkynes, addition of thiols to alkenes, addition of isothiocyanates to amines, and Diels-Alder cycloadditions. Importantly, because the starting materials for these reactions are relatively stable, in principle, they could be introduced to a wide range of macromolecules and hybrid materials. Furthermore, these reactions do not generate any by-products and operate on reasonable timescales (<12 hours), making them attractive for use in bioconjugation.
Biomedical research has benefited tremendously from peptide-based technologies because they have facilitated the elucidation of disease mechanisms and serve as novel and effective therapeutics. Specifically, an emerging theme in biotechnology is to use peptide variants to disrupt protein-protein interactions because compared to small molecules they have a larger surface area for binding, can recognize targets with higher specificity/affinity, and can be generated in weeks by phage display. Despite these successes and advantages, many peptides and most other large and charged biomolecules do not directly cross the cell plasma membrane to reach the cytosol without the aid of supporting transfection agents. Moreover, peptides are rapidly degraded in the biological milieu by proteases rendering them inactive. Identification of a potent p53/MDM2 inhibitor has been one of the central investigations in cancer research, where significant efforts have been undertaken to design small-molecule and peptide species capable of inhibiting this interaction in vivo. Several cell permeable small molecule compounds have been discovered, but they have suffered from serious off target effects and low intracellular activity. The peptide-based inhibitors failed, even the ones designed to penetrate cells, because they cannot enter cells effectively, despite high binding affinity and specificity. Thus a facile and reliable delivery of active and stable peptide-like molecules to the cytosol of cells is desirable.